Name: Radiochemical Assessment of Glycogen Synthase Enzyme Activity in Animal Tissue (Video) | JoVE Journal
Uploaded: 2025-10-24T14:50:00.000+00:00
Duration: 2 min 30 s
Description: Learn radiochemical assessment of glycogen synthase enzyme activity in murine skeletal muscle using genetically-engineered mouse models. This method enables direct measurement of metabolic enzyme activity with high sensitivity.

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5 minutes

Funding was provided in part by Ball State University, Indiana University School of Medicine-Muncie, and Aro Biotherapeutics.

All procedures were approved by the Ball State University Animal Care and Use Committee. Male and female cohorts of B6;129-Gaatm1Rabn/J&#160;wildtype mice, aged 3 months, are used as a representative example in this manuscript. The reagents and the equipment used are listed in the Table of Materials.
1. Preparation prior to assay
<ol>
	<li>Harvest tissue by cervical dislocation16 and immediately freeze in liquid N2 to preserve glycogen stores and store in liquid N2 or ultralow freezer until the start of the assay.</li>
	<li>Prepare the stock solutions prior to the start of the glycogen synthase assay (Table 1).</li>
	<li>Determine the number of replicate mouse samples and controls desired for the assay. Each mouse sample requires four total replicates, two for each reaction (presence or absence of G6P) (see Figure 1 and Table 2).</li>
	<li>Cut chromatography paper into 4 cm strips. Glycogen attaches to this paper and remains bound during the washing process. Using the width (6 mm) of a paper shredder, shred chromatography paper into thin individual segments.</li>
	<li>Using a pencil, label designated paper strips with mouse identification, blanks, and totals. Avoid using ink.</li>
	<li>Load chromatography papers into the wooden block template. Each template holds up to 12 papers, corresponding to one row of a 96-well plate.</li>
	<li>Obtain chromatography paper clamps (clipboard tops).</li>
	<li>Prepare one clamp for each row of reaction by clamping the edges of chromatography papers, leaving the majority of papers&#39; surface area exposed. Ensure labeled edges are visible.</li>
	<li>Label 1.7 mL microfuge tubes with sample identification. Two tubes per sample; one for homogenate and one for supernatant.</li>
	<li>Prior to the start of the assay, prepare both the + and - G6P reaction mixtures, see Table 3 for more information. Prepare 2,400 &#181;L aliquots of each reaction mixture.</li>
</ol>
2. Radiochemical glycogen synthase activity reaction
CAUTION: This step contains the use of [14C]-UDP-glucose, which is a radioactive material. Handle, store, and dispose of in a manner approved by the Nuclear Regulatory Commission (NRC).
<ol>
	<li>Add 30 &#181;L of [14C]-UDP-glucose (0.1 &#181;Ci/ &#181;L) to 2,400 &#181;L aliquots of both (+/- G6P) reaction mixes for a final concentration of 0.0012 &#181;Ci/ &#181;L. This recipe is suitable for 18 samples. 
	NOTE: The volume of [14C]-UDP-glucose can be changed to conserve usage and ensure adequate sensitivity. For example, use 50 &#181;L for mouse brain tissue and 25 &#181;L for mouse heart.</li>
	<li>Vortex reaction mixtures and aliquot 50 &#181;L to each well of a 96-well plate that will hold samples, blanks, and totals (Table 2). Afterward, cover with plate film and place at 4 C.</li>
	<li>Set out the protease inhibitors to thaw at room temperature. These inhibitors are itemized in Table 4 with additional information.</li>
	<li>Mass out 30-50 mg of frozen tissue. ~30 mg is optimal for skeletal muscle and heart samples; ~50 mg for brain. Keep tissue and microfuge tubes in liquid N2 and quickly weigh tissue to ensure the tissue stays frozen. Use a cooled pestle to grind tissue further.</li>
	<li>Prepare homogenization buffer with protease inhibitors on ice.</li>
	<li>One sample at a time, move the tube with tissue from liquid N2 to ice with a cold cap for 1 min. Add a cold cap (tube cap with three small holes cooled in liquid N2) to the sample tube while still in liquid N2. This prevents tissue loss as residual liquid N2 in the tube evaporates. Once out of liquid N2, samples should remain in ice.</li>
	<li>Once on ice, add volume (10-30 &#181;L/mg of tissue) of homogenization buffer to the sample and homogenize the sample with Tissue-Tearor. After completion of these two steps, move on to the next sample. 
	NOTE: Volume of buffer added to samples may be optimized depending on the source of tissue. For example: 30 &#181;L /mg for skeletal muscle, 15 &#181;L /mg for heart, and 10 &#181;L /mg for brain.</li>
	<li>Centrifuge samples for 5 min at 4 &#730;C at 3,600 g and transfer supernatant to a clean microfuge tube. Be careful not to touch the pellet. Vortex and add ~150 &#181;L to corresponding wells in a v-bottom 96-well plate on ice. Save the remainder for supernatant for Bradford assay (see step 2.15) 
	NOTE: If desired, additional sample aliquots may be saved for later assays such as immunoblotting. 
	CAUTION: Steps 2.9 through 2.14 require handling of 14C. Dispose of all contaminated materials in a manner approved by the NRC.</li>
	<li>Remove the reaction mix plate from 4 &#730;C and heat to 30 &#730;C on the thermocycler with the film removed. During this time, obtain 1 L of -20 &#730;C 66% ethanol and add to a wash beaker with a stir rod on&#160;stirring plate.</li>
	<li>Initiate reaction by adding 25 &#181;L of sample supernatant to reaction wells and 25 &#181;L of homogenization buffer to blank and total wells. Use a multichannel pipette to add to 1 row on the reaction plate at a time, waiting for at least 1 min between rows. Record the time that the reaction was initiated for each row. See Table 2 for more information. Use a multichannel pipette and ensure that equal volumes of liquid are added.</li>
	<li>Allow 10-15 min for the reaction to run; however, run time should be identical between rows.</li>
	<li>Aspirate 55 &#181;L from each well using a multichannel pipette and dispense onto the respective chromatography papers prepared previously. Mix the reaction by pipetting up and down before aspiration. Spot liquid in the center of the chromatography papers.</li>
	<li>Remove papers with totals from the clamp using forceps after spotting liquid from the well. These can be laid out on aluminum foil safely to dry. Drop all other chromatography papers into the stirring 66% ethanol bath. Glycogen is insoluble in 66% ethanol, while [14C]-UDP-glucose is soluble and will wash off chromatography papers.</li>
	<li>Repeat steps 2.11 and 2.12 for each row on the reaction plate. Ensure each row&#39;s reaction time is the same by recording the start and stop times of the reaction.</li>
	<li>Once all rows have been added to the stirring 66% ethanol, wash for 15 min. 
	NOTE: During this step and subsequent wash steps, sample aliquots may be prepared for complementary assays, such as the Bradford assay, responsible for determining the total volume of protein for normalization, which is necessary in calculating the enzymatic activity.</li>
	<li>Dispose of 66% ethanol without losing chromatography papers. Wash with 1 L of new 66% ethanol for 30 min. This 66% ethanol can be at room temperature.</li>
	<li>Repeat the previous step for a total of three 30 min 66% ethanol washes. 
	NOTE: If desired, the 66% ethanol from the final wash step can be saved and stored at -20 &#730;C for subsequent assays.</li>
	<li>After removal of 66% ethanol, submerge all chromatography papers in the wash beaker in acetone. Acetone facilitates the drying process.</li>
	<li>Remove chromatography papers from acetone and lay them on aluminum foil. Save or dispose of acetone.</li>
	<li>Dry chromatography papers under a heat lamp for 15 min. 
	NOTE: Dry papers may be stored indefinitely. Papers may be stored safely until ready to proceed to scintillation detection.</li>
</ol>
3. Measuring activity with scintillation detection
<ol>
	<li>Using forceps, move chromatography papers into scintillation vials held in scintillation racks. Record how the sample papers are oriented.</li>
	<li>Add 5 mL of scintillation fluid to each vial using the lid pump. Screw on caps for each vial.</li>
	<li>Load samples into the scintillation detector and load the halt rack behind the samples. Ensure the program for monitoring 14C is selected. Start detection by selecting Automatic counting.</li>
	<li>Print results</li>
	<li>Dispose of scintillation tubes and contents in a manner approved by the NRC.</li>
</ol>

<ol><li>Preiss, J., Walsh, D. A. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aPreiss%2C+J+%22Biology+of+Carbohydrates%22">Biology of Carbohydrates</a>. Ginsburg, V., Robbins, P. 1, John Wiley. 199-314 (1981).</li>
<li>Pederson, B. A. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aPederson%2C+BA+%22Enzymology+of+Complex+Alpha-Glucans%22">Enzymology of Complex Alpha-Glucans</a>. , CRC Press. 84-137 (2021).</li>
<li>Wayllace, N. Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+enzyme-coupled+continuous+spectrophotometric+assay+for+glycogen+synthases%5BTitle%5D)+AND+%22Mol+Biol+Rep%22%5BJournal%5D)">An enzyme-coupled continuous spectrophotometric assay for glycogen synthases.</a> Mol Biol Rep. 39 (1), 585-591 (2012).</li>
<li>Wilson, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Spectrophotometric+Methods+for+the+Study+of+Eukaryotic+Glycogen+Metabolism%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Spectrophotometric Methods for the Study of Eukaryotic Glycogen Metabolism.</a> J Vis Exp. (174), e63046(2021).</li>
<li>Danforth, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Glycogen+synthetase+activity+in+skeletal+muscle.+Interconversion+of+two+forms+and+control+of+glycogen+synthesis%5BTitle%5D)+AND+%22J+Biol+Chem%22%5BJournal%5D)">Glycogen synthetase activity in skeletal muscle. Interconversion of two forms and control of glycogen synthesis.</a> J Biol Chem. 240, 588-593 (1965).</li>
<li>Leloir, L. F., Olavarria, J. M., Goldemberg, S. H., Carminatti, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Biosynthesis+of+glycogen+from+uridine+diphosphate+glucose%5BTitle%5D)+AND+%22Arch+Biochem+Biophys%22%5BJournal%5D)">Biosynthesis of glycogen from uridine diphosphate glucose.</a> Arch Biochem Biophys. 81 (2), 508-520 (1959).</li>
<li>Schalin-Jantti, C., Harkonen, M., Groop, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Impaired+activation+of+glycogen+synthase+in+people+at+increased+risk+for+developing+NIDDM%5BTitle%5D)+AND+%22Diabetes%22%5BJournal%5D)">Impaired activation of glycogen synthase in people at increased risk for developing NIDDM.</a> Diabetes. 41 (5), 598-604 (1992).</li>
<li>Huang, X., Hansson, M., Laurila, E., Ahren, B., Groop, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fat+feeding+impairs+glycogen+synthase+activity+in+mice+without+effects+on+its+gene+expression%5BTitle%5D)+AND+%22Metabolism%22%5BJournal%5D)">Fat feeding impairs glycogen synthase activity in mice without effects on its gene expression.</a> Metabolism. 52 (5), 535-539 (2003).</li>
<li>Holt, B. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+novel+CD71+Centyrin%3A+Gys1+siRNA+conjugate+reduces+glycogen+synthesis+and+glycogen+levels+in+a+mouse+model+of+Pompe+disease%5BTitle%5D)+AND+%22Mol+Ther%22%5BJournal%5D)">A novel CD71 Centyrin: Gys1 siRNA conjugate reduces glycogen synthesis and glycogen levels in a mouse model of Pompe disease.</a> Mol Ther. 33 (1), 235-248 (2025).</li>
<li>Prats, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Phosphorylation-dependent+translocation+of+glycogen+synthase+to+a+novel+structure+during+glycogen+resynthesis%5BTitle%5D)+AND+%22J+Biol+Chem%22%5BJournal%5D)">Phosphorylation-dependent translocation of glycogen synthase to a novel structure during glycogen resynthesis.</a> J Biol Chem. 280 (24), 23165-23172 (2005).</li>
<li>Perezleon, J. A., Osorio-Paz, I., Francois, L., Salceda, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Immunohistochemical+localization+of+glycogen+synthase+and+GSK3beta%3A+Control+of+glycogen+content+in+retina%5BTitle%5D)+AND+%22Neurochem+Res%22%5BJournal%5D)">Immunohistochemical localization of glycogen synthase and GSK3beta: Control of glycogen content in retina.</a> Neurochem Res. 38 (5), 1063-1069 (2013).</li>
<li>Thomas, J. A., Schlender, K. K., Larner, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+rapid+filter+paper+assay+for+UDP-glucose-glycogen+glucosyltransferase%2C+including+an+improved+biosynthesis+of+UDP-14C-glucose%5BTitle%5D)+AND+%22Analytical+Biochemistry%22%5BJournal%5D)">A rapid filter paper assay for UDP-glucose-glycogen glucosyltransferase, including an improved biosynthesis of UDP-14C-glucose.</a> Analytical Biochemistry. 25 (1), 486-499 (1968).</li>
<li>Parker, G. J., Lund, K. C., Taylor, R. P., McClain, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Insulin+resistance+of+glycogen+synthase+mediated+by+O-linked+N-acetylglucosamine%5BTitle%5D)+AND+%22J+Biol+Chem%22%5BJournal%5D)">Insulin resistance of glycogen synthase mediated by O-linked N-acetylglucosamine.</a> J Biol Chem. 278 (12), 10022-10027 (2003).</li>
<li>Niederwanger, A., Kranebitter, M., Ritsch, A., Patsch, J. R., Pedrini, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+gel+filtration+assay+to+determine+glycogen+synthase+activity%5BTitle%5D)+AND+%22J+Chromatogr+B+Analyt+Technol+Biomed+Life+Sci%22%5BJournal%5D)">A gel filtration assay to determine glycogen synthase activity.</a> J Chromatogr B Analyt Technol Biomed Life Sci. 820 (1), 143-145 (2005).</li>
<li>Suzuki, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Insulin+control+of+glycogen+metabolism+in+knockout+mice+lacking+the+muscle-specific+protein+phosphatase+PP1G%2FRGL%5BTitle%5D)+AND+%22Mol+Cell+Biol%22%5BJournal%5D)">Insulin control of glycogen metabolism in knockout mice lacking the muscle-specific protein phosphatase PP1G/RGL.</a> Mol Cell Biol. 21 (8), 2683-2694 (2001).</li>
<li>Nagy, A., Gertsenstein, M., Vintersten, K., Behringer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quick+and+humane+sacrifice+of+a+mouse+by+cervical+dislocation%5BTitle%5D)+AND+%22CSH+Protoc%22%5BJournal%5D)">Quick and humane sacrifice of a mouse by cervical dislocation.</a> CSH Protoc. 2006 (1), prot4357(2006).</li>
<li>Gomes, F. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Glycogen+storage+and+muscle+glucose+transporters+%28GLUT-4%29+of+mice+selectively+bred+for+high+voluntary+wheel+running%5BTitle%5D)+AND+%22J+Exp+Biol%22%5BJournal%5D)">Glycogen storage and muscle glucose transporters (GLUT-4) of mice selectively bred for high voluntary wheel running.</a> J Exp Biol. 212 (Pt 2), 238-248 (2009).</li>
<li>Greenberg, C. C., Jurczak, M. J., Danos, A. M., Brady, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Glycogen+branches+out%3A+new+perspectives+on+the+role+of+glycogen+metabolism+in+the+integration+of+metabolic+pathways%5BTitle%5D)+AND+%22Am+J+Physiol+Endocrinol+Metab%22%5BJournal%5D)">Glycogen branches out: new perspectives on the role of glycogen metabolism in the integration of metabolic pathways.</a> Am J Physiol Endocrinol Metab. 291 (1), E1-E8 (2006).</li>
<li>Bradford, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+rapid+and+sensitive+method+for+the+quantitation+of+microgram+quantities+of+protein+utilizing+the+principle+of+protein-dye+binding%5BTitle%5D)+AND+%22Anal+Biochem%22%5BJournal%5D)">A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.</a> Anal Biochem. 72, 248-254 (1976).</li>
<li>Gilboe, D. P., Larson, K. L., Nuttall, F. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Radioactive+method+for+the+assay+of+glycogen+phosphorylases%5BTitle%5D)+AND+%22Anal+Biochem%22%5BJournal%5D)">Radioactive method for the assay of glycogen phosphorylases.</a> Anal Biochem. 47 (1), 20-27 (1972).</li>
</ol>

The authors have nothing to disclose.

Measurement of glycogen synthase enzyme activity is a valuable tool for monitoring glycogen metabolism in various metabolic and disease states. Measurement in the absence and presence of glucose-6-phosphate (G6P) offers insight into the regulatory status of the enzyme and an indirect index of the phosphorylation state. [U-14C]-UDP-glucose incorporation assay is an efficient and well-documented technique for measuring glycogen synthase enzymatic activity, offering high sensitivity, substrate specificity, and ad...

Glycogen, a highly branched polysaccharide composed of glucose units, serves as an important energy reserve in various tissues, including muscles, liver, and brain1. Its synthesis and degradation are orchestrated by a complex system of specialized enzymes and regulatory proteins. Vital to its biosynthesis is the enzyme glycogen synthase (GYS), of which there are two isoforms (GYS1 and GYS2). GYS1 is a regulatory enzyme directly responsible for the creation of glycogen from glucose in non-liver cells, while GYS2 functions in the liver. This process, known as glycogenesis, is the sequential addition of an individual unit of glucose onto a growing glycogen chain. GYS catalyzes the transfer of glucose from the intermediate uridine diphosphate glucose (UDP-glucose) to the end of a glycogen chain, creating an alpha-1,4-glycosidic linkage2. In coordination with the glycogen branching enzyme, this reaction extends glycogen molecules, providing both efficient energy storage and release.
Assay techniques exist to measure the activity of the enzyme, with one of the more common groupings of methods consisting of those of the spectrophotometric variety3,4. These assays typically rely on a system of indirect coupled reactions and measure the absorbance of the downstream product, NADH, at 340 nm. One unit of NADH is consumed&#160;for every unit of UDP freed from UDP-glucose by glycogen synthase, producing a measurable excitation that directly correlates to glycogen synthase activity5,6. While spectrophotometry assays are relatively inexpensive and accessible, they come with the minor downside of relying on an indirect reaction and major downsides of interference by sample turbidity and lower sensitivity, especially an issue for smaller samples or samples with little/highly phosphorylated glycogen synthase. Fluorometric assays, using the coupled reactions described above, have been developed to improve sensitivity in muscle tissue from human7 and mouse8. Other techniques exist for quantifying glycogen synthase, such as immunoblotting9, and immunohistochemistry10; however, these techniques are only able to quantify the physical amount of enzyme and, at best, can infer activity from phosphorylation but not directly quantify enzyme activity11.
Among these enzyme assay techniques, radiochemical methods remain the most widely used for determining glycogen synthase activity in mammalian tissue due to their lack of interference by sample turbidity and their high sensitivity8,9. These assays incorporate radiolabeled glucose from UDP-glucose labeled with either 14C12 or 3H13 into glycogen, allowing for direct measurement of enzyme activity over a set amount of time. In the following procedure, a tissue sample containing glycogen synthase is exposed to UDP-glucose and glycogen primer. Glycogen synthase catalyzes the reaction shown in the equation below:
UDP-[14C]glucose + (glycogen)n &#8594; (glycogen)n+1[14C] + UDP
After a set amount of time, the reaction is terminated and the glycogen containing radiolabeled glucose is either precipitated onto filter paper12 (as described below) or subjected to gel filtration as described by Niederwanger et al.14 and measured using a scintillation detector. This method also includes a separate identical reaction that includes glucose-6-phosphate (G6P), an allosteric activator of glycogen synthase. Under conditions with sufficient levels of G6P, glycogen synthase activity is maximally activated, showcasing the absolute potential for glycogen synthase activity of a given sample. The method described here was modified from Suzuki and colleagues15 to a 96-well format and is optimized for use in mouse muscle tissue; however, the protocol can be altered for other tissues and animal species.
Despite the inherent cost and safety considerations associated with radioactive materials, radiochemical assays offer significant advantages over non-radioactive alternatives, provided instruments are available. The following procedure details a highly sensitive and robust means of determining the activity of glycogen synthase, a key enzyme in glycogen metabolism, in animal muscle tissue.

<table><tbody><tr><td>[&lt;sup&gt;14&lt;/sup&gt;C] Uridine 5-diphospho-glucose</td><td>American Radiolabeled Chemicals</td><td>ARC 0154</td><td></td></tr><tr><td>31 ETCHR chromatography paper</td><td>Whatman</td><td>3031 915</td><td></td></tr><tr><td>Benzamidine hydrochloride hydrate</td><td>Sigma</td><td>B6506</td><td></td></tr><tr><td>Beta mercaptoethanol</td><td>Sigma</td><td>M3148</td><td></td></tr><tr><td>Dry bath</td><td>Torey Pines Scientific</td><td>SC25</td><td></td></tr><tr><td>Ethanol (200 proof)</td><td>PharmcoAaper</td><td>111000200</td><td></td></tr><tr><td>Ethylene glycol tetraacetic acid (EGTA)</td><td>Sigma</td><td>E4378</td><td></td></tr><tr><td>Ethylenediaminetetraacetic acid (EDTA)</td><td>Sigma</td><td>E5134</td><td></td></tr><tr><td>Glucose-6-phosphate sodium salt</td><td>Sigma</td><td>G7879</td><td></td></tr><tr><td>Glycogen from rabbit liver (Type III)</td><td>Sigma</td><td>G8876</td><td></td></tr><tr><td>Microplate, flat-bottom, 96 well</td><td>Greiner</td><td>655101</td><td></td></tr><tr><td>Microplate, pcr, 96 well</td><td>DOT Scientific</td><td>951-PCR</td><td></td></tr><tr><td>Multi-Purpose Scintillation Counter</td><td>Beckman Coulter</td><td>LS 6500</td><td></td></tr><tr><td>Potassium fluoride dihydrate</td><td>Sigma</td><td>221872</td><td></td></tr><tr><td>Protease inhibitor cocktail</td><td>GoldBio</td><td>GB-108-2</td><td></td></tr><tr><td>Scintillation cocktail</td><td>Reasearch Products International</td><td>111195</td><td></td></tr><tr><td>Scintillation vials</td><td>Reasearch Products International</td><td>125509</td><td></td></tr><tr><td>Sodium fluoride</td><td>Sigma</td><td>S7920</td><td></td></tr><tr><td>Tissue Tearor</td><td>Biospec Products</td><td>Model 398</td><td></td></tr><tr><td>Tosyllysine Chloromethyl Ketone (TLCK)</td><td>Sigma</td><td>T7254</td><td></td></tr><tr><td>Trizma base (99.9%)</td><td>Sigma</td><td>T1503</td><td></td></tr><tr><td>Trizma hydrochloride</td><td>Sigma</td><td>T3253</td><td></td></tr><tr><td>Uridine 5-diphospo-glucose</td><td>Sigma</td><td>U-4625</td><td></td></tr></tbody></table>

radiochemical assessment of glycogen synthase enzyme activity in animal tissue

Radiochemical Assessment of Glycogen Synthase Enzyme Activity in Animal Tissue

Glycogen is an important metabolite to many eukaryotic cells, integral to energy storage and quick energy release. The building of the molecule is dependent on glycogen synthase, a highly regulated enzyme with the primary function of facilitating the sequential attachment of glucose units in a growing glycogen polymer. This enzyme is allosterically activated by glucose-6-phosphate and inhibited at multiple sites by phosphorylation. Knowledge of glycogen synthase enzymatic activity is a key component of understanding glycogen metabolism, muscle energy levels, and metabolic disorders such as diabetes and glycogen storage diseases. Described here is a highly sensitive and effective method of determining the activity of glycogen synthase in animal muscle tissue by measuring the amount of integrated glucose, from radio-labeled [14C]-UDP-glucose, into glycogen molecules. This procedure utilizes radioactive 14C and will require the proper authorization and lab space for use and disposal. This procedure will also require access to a liquid scintillation counter.

Following the protocol, a printout created from the scintillation detector will contain the measured amount of ionizing radiation emitted from each chromatography paper containing vial, directly correlated to the activity level of glycogen synthase. The scintillation detector will compute the counts per minute (CPM) of detected radiation for each sample, blank, and total over a set amount of time. First convert CPM to DPM (disintegrations per min), which is based on the efficiency of the scintillation counter used (DPM=C...

The animal tissue assay utilizes nuclear scintillation detection to determine the enzymatic activity of glycogen synthase by measuring the incorporation of radio-labeled glucose into glycogen.

Glycogen is an important metabolite to many eukaryotic cells, integral to energy storage and quick energy release. The building of the molecule is ...

Our research investigates glycogen metabolism, particularly glycogen synthase in health and disease, using genetically-engineered mouse models. Research on glycogen storage diseases primarily centers on developing treatments that minimize buildup of toxic glycogen. Begin by taking the prepared murine skeletal muscle homogenate and the 14-carbon UDP-glucose reaction mix plate.Using a multi-channel pipette, add 25 microliters of the sample supernatant to the designated reaction wells. Add 25 microliters of homogenization buffer to the blank and total wells. Record the exact time at which the reaction is initiated for each row.Ensure that equal volumes are added across all wells. Afterward, mix the reaction in each well by pipetting up and down several times. Using a multi-channel pipette, aspirate 55 microliters from each well and dispense the liquid onto the corresponding chromatography papers prepared earlier.Spot the liquid carefully at the center of each paper. Using forceps, remove the chromatography papers labeled as totals from the clamp after spotting the liquid. Place these total papers on aluminum foil to air-dry safely.Drop all other chromatography papers into a stirring 66%ethanol bath. Quadriceps glycogen synthase activity in murine skeletal muscle was lower under basal conditions without glucose 6-phosphate and significantly higher under maximal conditions with glucose 6-phosphate, consistent with partial phosphorylation of glycogen synthase. This protocol describing high throughput assessment of tissue glycogen synthase enzymatic activity using radioisotopes currently has no published protocol.Protocol advantages are a direct measurement of enzyme activity and high sensitivity, integral for use on samples with low enzyme activity.

radiochemical-assessment-glycogen-synthase-enzyme-activity-animal

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Determination of the Glycogen Content in Cyanobacteria

Intact regulation of muscle glucose uptake is important for maintaining whole body glucose homeostasis. This protocol presents assessment of insulin- and contraction-stimulated glucose uptake in isolated and incubated mature skeletal muscle when delineating the impact of various physiological interventions on whole body glucose metabolism.

Measurement of Insulin- and Contraction-Stimulated Glucose Uptake in Isolated and Incubated Mature Skeletal Muscle from Mice

Techniques to measure the activity of key enzymes of glycogen metabolism are presented, using a simple spectrophotometer operating in the visible range.

Spectrophotometric Methods for the Study of Eukaryotic Glycogen Metabolism

An optimal sucrose concentration was determined for the extraction of liver glycogen using sucrose density gradient centrifugation. The addition of a 10 min boiling step to inhibit glycogen-degrading enzymes proved beneficial.

The Extraction of Liver Glycogen Molecules for Glycogen Structure Determination

A modified post-fixation procedure increases the contrast of glycogen particles in tissue. This paper provides a step-by-step protocol describing how to handle the tissue, conduct the imaging, and use stereological methods to obtain unbiased and quantitative data on fiber type-specific subcellular glycogen distribution in skeletal muscle.

Quantification of Subcellular Glycogen Distribution in Skeletal Muscle Fibers using Transmission Electron Microscopy

As mitochondria are only a small percentage of the plant cell, they need to be purified for a range of studies. Mitochondria can be isolated from a variety of plant organs by homogenization, followed by differential and density gradient centrifugation to obtain a highly purified mitochondrial fraction.

Isolation and Respiratory Measurements of Mitochondria from Arabidopsis thaliana

Learn radiochemical assessment of glycogen synthase enzyme activity in murine skeletal muscle using genetically-engineered mouse models. This method enables direct measurement of metabolic enzyme activity with high sensitivity.

Radiochemical Assessment of Glycogen Synthase Enzyme Activity in Animal Tissue

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